EXCHANGE 


1919?! 


Fat  Associated  With  Starch 


By 
T.  CLINTON  TAYLOR,  Chem.E.,  A.KL 


SUBMITTED  IN  PARTIAL  FULFILMENT  OP  THE  REQUIREMENTS  FOR  THE 

DEGREE  OF  DOCTOR  OF  PHILOSOPHY  IN  THE  FACULTY 

OF  PURE  SCIENCE,  COLUMBIA  UNIVERSITY 


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COLUMBIA  UNIVERSITY  PRESS 

1920 
All  rights  reserved 


Fat  Associated  With  Starch 


By 
T.  CLINTON  TAYLOR,  ChemJE.,  A.M. 


SUBMITTED  IN  PARTIAL  FULFILMENT  OF  THE  REQUIREMENTS  FOR  THE 

DEGREE  OF  DOCTOR  OF  PHILOSOPHY  IN  THE  FACULTY 

OF  PURE  SCIENCE,  COLUMBIA  UNIVERSITY 


COLUMBIA  UNIVERSITY  PRESS 

1920 
All  rights  resened 


Copyright,  1920 
By  COLUMBIA  UNIVERSITY  PRESS 


Printed  from  type,  December,  1920 


TO  MY  FATHER  AND  MOTHER 


451.733 


ACKNOWLEDGMENT 

The  author  wishes  to  express,  his  gratitude  to  Professor  J.  M.  Nelson 
for  his  many  suggestions,  his  advice  and  encouragement  throughout  the 
work.  T.  C.  T. 


ABSTRACT  OF  DISSERTATION. 

1.  What  was  attempted? 

2.  In  how  far  were  the  attempts  successful? 

3.  What  contribution  actually  new  to  the  science  of  chemistry  has  been 
made? 

1.  In  the  production  of  glucose  by  the  hydrolysis  of  starch,  a  consider- 
able quantity  of  fat  occurs  in  the  glucose  liquor.     From  the  previous 
treatment  of  the  raw  starch  this  fat  appears  to  be  an  inherent  part  of  the 
starch  itself.     This  investigation  was  undertaken  to  determine  whether 
fat  is  really  combined  with  the  carbohydrate  in  the  starch  molecule. 

2.  It  was  shown  that: 

(a)  Fat  is  liberated  upon  the  hydrolysis  of  starch. 

(6)  Although  no  definite  compound  of  carbohydrate  and  fat  was  iso- 
lated in  pure  condition,  still  a  residue  relatively  high  in  fat  content  was 
obtained. 

(c)  The  fatty  acid  of  the  fat  was  chiefly  palmitic. 

(d)  The  palmitic  acid  was  combined  with  an  unsaturated  material,  of 
unknown  composition,  as  an  ester. 

0)  The  unsaturated  material  served  as  a  link  between  the  palmitic 
acid  and  the  carbohydrate  part  of  the  starch. 

3.  It  has  been  shown  that  maize  starch  cannot  be  regarded  as  simply 
made  up  of  glucose,  but  contains  a  minor  constituent,  a  fatty  substance 
made  up  of  palmitic  acid  and  an  unsaturated  material  whose  composition 
is  still  unknown. 


FAT  ASSOCIATED  WITH  STARCH. 

It  is  well  known  that  the  starches  as  they  are  obtained  ordinarily  from 
the  plants  have  a  certain  amount  of  fat  associated  with  them.  It  has 
been  assumed  generally  that  this  fat  can  be  removed  by  solvents,  and 
that  its  presence  is  a  contamination  of  the  starch  with  other  constituents 
which  occur  intimately  associated  with  the  starch  in  the  plant. 

In  the  manufacture  of  glucose  commercially  from  corn  starch  an 
insoluble  product  known  as  "refinery  mud"  occurs  in  the  sugar  liquor 
after  the  hydrolysis,  and  is  separated  by  filtration.  This  refinery  mud  as 
recovered  contains  about  50%  of  fat,  chiefly  the  higher  fatty  acids,  and 
after  washing  and  other  treatment  is  sold  as  soap  stock. 

Considering  the  process  employed  in  the  commercial  hydrolysis  of 
starch  it  is  improbable  that  the  source  of  this  fatty  material  can  be  at- 
tributed to  extraneous  matter  accompanying  the  starch  from  the  kernel, 
since  its  presence  is  observed  only  during  or  after  the  destruction  of  the 
starch  by  hydrolysis.  The  occurrence  of  free  fatty  acids  during  the 
hydrolysis  and  disruption  of  the  complex  starch  molecule  leads  to  the 
interesting  question  of  whether  the  fatty  material  constitutes  an  inherent 
part  of  the  starch  itself. 

The  present  article  is  an  account  of  an  attempt  to  answer  this  question, 
and  the  results  obtained  lead  to  the  following  conclusions : 

I.  The  major  part  of  the  fatty  material  present  in  starch  cannot  be 
removed  by  solvents  before  hydrolysis. 

II.  Hydrolysis  of  corn  starch  freed  of  extraneous  fat  liberates  fatty 
acids. 

III.  The  liberated  fat  is  principally  palmitic  acid,  but  an  unsaturated 
substance  of  unknown  structure  also  occurs  with  it. 

IV.  The  fat  is  liberated  when  hydrolysis  has  reached  the  erythrodextrin 
stage. 

V.  It  is  possible  to  obtain  from  starch  residues  containing  relatively 
large  amounts  of  fat  combined  with  carbohydrate. 

VI.  The  palmitic  acid  apparently  is  attached  indirectly  to  the  carbo- 
hydrate, but  directly  to  the  unsaturated  component. 

VII.  Starches  from  other  sources  than  corn  also  contain  combined  fat. 

I.  Extraneous  Fat  Accompanying  the  Starch. 

The  corn  starch  used  was  the  best  alkali-washed  product  available 
in  the  open  market.  A  82.26  g.  sample  of  this  material  was  extracted, 
first  with  ether,  then  with  petroleum  ether  and  finally  with  carbon  tetra- 


chloride.    The  duration  of  the  extraction  in  each  case  was  36  hours. 

The  amount  of  soluble  matter,  chiefly  fat,  obtained  by  means  of  each 
successive  solvent  was 

Solvent.  %. 

Ether  (dry) 0.057 

Petroleum  ether 0.012 

Carbon  tetrachloride 0.046 


Total 0.115 

Further  extraction  gave  no  weighable  residues  upon  evaporation  of  the 
solvent. 

The  residues,  obtained  by  evaporation  of  the  solutions  from  the  first 
extractions  and  the  weights  of  which  are  given  in  the  table  above,  were 
yellow  and  gummy.  Upon  solution  in  alcohol  and  titration  with  alkali, 
the  combined  residues  gave  an  acid  value1  of  95.1.  A  comparison  of  this 
acid  number  with  186.0,  that  of  the  fat  described  under  II,  indicates  the 
presence  of  considerable  foreign  matter,  probably  nitrogeneous  in  character. 

Purification  of  the  Corn  Starch. — Since  starch  is  separated  me- 
chanically from  the  other  substances  which  occur  with  it  in  the  plant,2 
the  separation  is  never  complete,  even  after  repeated  treatments.  For 
this  reason,  it  is  necessary  to  resort  to  other  methods  of  removing  the 
remaining  impurities,3  chiefly  nitrogeneous.  The  presence  of  the  latter 
is  indicated  both  by  the  nitrogen  content  0.06%  (Kjeldahl  method),  and 
the  presence  pf  dark  yellow  particles  when  the  starch,  stained  with  iodine, 
is  examined  under  the  microscope. 

Removal  of  the  Nitrogenous  Material  and  Extraneous  Fat. — About 
500  g.  of  dry  powdered  corn  starch  was  placed  hi  a  3-liter  round-bottom 
flask  provided  with  a  reflux  condenser,  then  enough  95%  alcohol  added 
to  make  a  thick  paste  and  finally  500  cc.  of  90%  alcohol  containing  0.23 
g.  of  hydrogen  chloride  per  cc.  After  refluxing  on  the  water  bath  for 
3/4  hour  with  continuous  agitation,  the  contents  of  the  flask  was  filtered 
through  a  Buchner  funnel  and  the  residue  washed,  first  with  hot  95% 
alcohol  and  finally  with  several  portions  of  ether. 

The  purified  starch  obtained  in  this  way  was  clean  and  white.  When 
stained  with  iodine,  it  showed  under  the  microscope  no  indications  of 
protein,  and  the  granules  appeared  to  be  still  intact,  although  slightly 
enlarged,  with  evidence  of  incipient  rupture  at  the  hilum.  That  no 
profound  change  in  the  starch  had  occurred  was  shown  also  by  the  fact 
that  when  a  sample  of  this  starch,  treated  with  alkali  to  neutralize  the 
hydrochloric  acid  which  still  adhered,  was  gelatinized  by  stirring  into 

1  Mg.  of  potassium  hydroxide  neutralized  by  one  g.  of  the  fat. 
8  Commercial  method,  T.  B.  Wagner,  J.  Soc.  Chem.  Ind.,  28,  343  (1909). 
1  Composition  of  corn  kernel,  J.  Am.  Chem.  Soc.  25,  1166  (1903);  and  Porst,  Eighth 
Inter.  Cong.  A  pp.  Chem.,  12,  205. 


boiling  water  it  gave  a  "thick  boiling"  paste  which  is  characteristic  of 
unchanged  or  "raw"  starch.  The  amount  of  hydrochloric  acid  adhering 
was  determined  by  drying  the  purified  starch  at  40°  and  then  suspending 
in  water  and  titrating  with  standard  alkali,  and  was  found  to  be  0.52%. 
By  means  of  Kjeldahl  determinations,  the  nitrogen  content  was  found 
to  have  been  decreased  in  the  process  of  purification  from 0.06  to  o  04%. 

When  the  purified  starch  was  stained  with  iodine  and  examined  under  the 
microscope,  a  stratified  structure  of  alternating  dark  blue  and  transparent 
concentric  rings  was  observed.  This  is  in  accord  with  previous  observa- 
tions of  Duclaux1  and  others  and  indicates  a  layer  structure  of  the  starch 
granules,  which  might  be  of  importance  in  connection  with  the  question 
of  the  presence  of  2  kinds  of  starch  material  in  the  granules,  as  held  by 
Baker  and  Sherman  and  others.2 

The  starch  purified  by  the  latter  method  contains  no  extraneous  fat. 
Eighty  g.  of  dried  starch  was  extracted  in  a  Soxhlet  for  40  hours  with  dry 
ether.  Upon  evaporation  of  the  ether  extract  no  weighable  residue  re- 
mained, showing  the  starch  to  be  free  from  extraneous  fat. 

In  order  to  make  sure  that  no  free  fat  was  adsorbed  so  firmly  to  the 
starch  that  it  could  not  be  removed  by  the  above  method  of  extraction 
the  following  procedure  was  employed. 

An  ether  solution  of  fat,  previously  obtained  from  hydrolyzed  starch, 
was  thoroughly  mixed  with  finely  powdered  purified  starch  and  this 
mixture,  containing  the  starch  in  suspension,  evaporated,  then  heated 
on  the  water  bath  with  stirring.  The  fat-impregnated  starch  was  then 
extracted  for  10  hours  in  a  Soxhlet  with  absolute  ether  and  the  amount 
of  fat  determined. 

SAMPLE,  20.0  G. 

Fat  added.  Fat  recovered 

G.  G. 

1 0.4566  0.4501 

2 0.4035  0.4057 

3 0. 1400  0. 1465 

In  Numbers  i  and  2  the  fat  added  was  much  in  excess  of  total  fat 
usually  found,  while  in  3  the  amount  was  comparable  with  the  usual  fat 
content  of  the  original  starch. 

On  hydrolysis  of  these  starch  samples  with  aqueous  hydrochloric  acid, 
after  the  added  fat  had  been  removed  by  extraction,  the  usual  amount  of 
fatty  material  was  recovered,  as  in  II. 

II.  Liberation  of  Fatty  Acids. 

When  the  starch  freed  from  extraneous  fat  as  described  in  I  is  hy- 
drolyzed, fat  is  liberated,  exceeding  in  amount  the  extraneous  fat  ex- 
tracted from  the  original  starch. 

1  Duclaux,  Ann.  Inst,  Pasteur,  9,  836  (1895). 
1  J.  Am.  Chem.  Soc.,  38,  (1916). 


IO 

Fat  by  Hydrolysis. — One  hundred  cc.  of  cone,  hydrochloric  acid  was 
added  to  200  cc.  of  boiling  water,  and  immediately  upon  addition  of  the 
acid  a  starch  suspension  of  25  g.  of  starch  in  100  cc.  of  water  was  poured 
slowly  into  the  boiling  acid  solution  with  continuous  stirring  and  heating. 
The  mixture  soon  liquefied  and  the  starch  was  hydrolyzed  with  the  libera- 
tion of  an  insoluble,  flocculent  material.  The  heating  was  discontinued 
when  a  drop  of  the  liquid  no  longer  showed  color  with  iodine,  and  the 
mixture  was  allowed  to  cool.  It  was  then  poured  on  a  large  filter  paper, 
allowed  to  drain,  and  the  residue  washed  with  cold  water  until  free  from 
hydrochloric  acid.  The  paper  and  residue  were  dried  at  50°  in  an  air- 
bath,  placed  in  a  thimble  and  extracted  in  a  Soxhlet  for  3  hours.  After 
removal  of  the  solvent  from  the  extract,  by  evaporation  on  the  steam- 
bath,  the  flask  and  the  remaining  residue  were  dried  and  weighed. 

Both  ethyl  ether  and  petroleum  ether  were  used  in  the  above.  The 
former  seemed  to  give  slightly  higher  results  and  always  carried  with  it 
more  coloring  matter,  besides  a  small  amount  of  reducing  sugar,  while  the 
latter  gave  a  cleaner  product  but  it  was  found  difficult  to  get  rid  of  the 
last  traces  of  the  solvent. 

Four  samples  of  purified  starch  were  subjected  to  the  above  treatment 
and  the  fat  liberated  by  hydrolysis  determined  and  given  below. 

SAMPLE,  25  G.  OP  STARCH. 

Fat  by  Acid 

Solvent.  Extraction.         hydrolysis.        number. 

% 

i - Petroleum  ether  Dry  o .  56  181.3 

2 Petroleum  ether  Dry  o .  49  186 . 5 

3 Petroleum  ether  Wet  o. 54 

4 Ethyl  ether  Dry  o. 64  184 . 7 

Wet  extraction  with  ethyl  ether  of  the  hydrolyzed  starch  liquor  necessi- 
tated drying  the  ether  layer  due  to  the  relatively  large  amount  of  water 
taken  up  by  it.  As  this  entails  some  loss,  and  further  since  in  the  presence 
of  fat,  reducing  sugar  passes  into  the  ether  layer,  only  extraction  of  dried 
fat  should  be  attempted  with  this  solvent. 

The  experiment  corresponding  to  the  data  marked  3  in  the  above  table 
was  carried  out  slightly  differently,  in  that  the  liquor  resulting  from  the 
hydrolysis  was  extracted  directly  with  petroleum  ether,  without  first 
filtering  or  drying  the  residue. 

The  fat  obtained  by  the  hydrolysis  of  the  starch  will  hereafter  be 
designated  as  "fat  by  hydrolysis"  to  distinguish  it  from  the  small  amount 
of  extraneous  fat,  extracted  directly  from  the  starch. 

Gelatinization  and  destruction  of  starch  granule  does  not  affect  com- 
bined fat.  In  order  to  make  sure  that  none  of  the  fat  obtained  by  the 
hydrolysis  of  the  starch,  the  amounts  of  which  are  given  in  the  table  in 


II 

Part  II,  was  held  mechanically  as  extraneous  fat  between  the  layers  in  the 
starch  granule,  the  latter  were  destroyed  by  gelatinization.  One  hundred 
parts  of  dry  powdered  starch  were  added  to  80  parts  of  a  50%  aqueous 
solution  of  ammonium  thiocyanate  to  which  had  been  added  previously 
40%  of  its  weight  of  alcohol.1  The  gelatinized  paste  thus  obtained  was 
stirred  with  an  excess  of  acetone  which  precipitated  a  white  amorphous 
starch  containing  no  granules.  Practically  all  of  the  ammonium  thio- 
cyanate was  then  removed  by  repeated  washing  with  acetone  and  alcohol 
and  the  dried  product  finally  extracted  with  ether.  A  sample  of  this 
ether-washed  starch  contained  no  directly  extractable  fat,  but  did  contain 
0.41%  of  "fat  by  hydrolysis"  and  the  latter  had  an  acid  number  of  182.7. 
Since  the  amount  of  fat  and  its  acid  number  agree  very  well  with  the 
values  in  the  above  table  for  starch  in  the  form  of  granules,  it  is  evident 
that  the  "fat  by  hydrolysis"  cannot  be  attributed  to  retention  of 
fat  between  the  layers  in  the  granules.  Had  this  been  the  case  then 
when  the  granules  were  destroyed  the  fat  would  have  been  removed 
by  the  acetone  and  alcohol,  and  the  fat  obtained  upon  subsequent 
hydrolysis  of  the  amorphous  material  would  have  been  much  lower 
than  0.41%. 

III.  Nature  of  the  Fatty  Material  Liberated  by  the  Hydrolysis  of  Maize 

Starch. 

The  fatty  material  obtained  by  the  hydrolysis  of  the  starch  as  de- 
scribed under  II,  is  semi-crystalline,  forms  soap  with  alkali,  produces 
grease  stains  on  paper  and  has  a  "tallow" -like  odor  which  becomes  more 
pronounced  on  heating. 

In  order  to  obtain  more  definite  data  as  to  the  exact  nature  of  the  fatty 
material,  a  much  larger  quantity  was  necessary  than  could  be  obtained 
by  the  hydrolysis  of  batches  of  starch  in  ordinary  chemical  laboratory 
apparatus.  Therefore,  the  "refinery  mud" .  mentioned  in  Part  I  was 
used.2  This  mud  corresponds  to  the  flocculent  precipitate  which  remains 
after  acid  hydrolysis  of  the  starch  in  the  method  for  "fat  by  hydrolysis." 
The  commercial  production  of  glucose  from  starch  is  simply  this  aqueous 
acid  hydrolysis  modified  to  fit  large  scale  production. 

Due  to  the  fact  that  the  mud  is  removed  by  filtration  in  iron  filter 
presses,  a  considerable  amount  of  iron  soap  is  formed  from  the  action 
of  the  fatty  acids  on  the  apparatus.  A  proximate  analysis  of  this  "re- 
finery mud"  shows  the  following: 

1  /.  Soc.  Chem.  Ind.,  28,  213  (1909). 

8  The  authors  wish  to  thank  Dr.  C.  E.  G.  Porst  for  procuring  from  the  Corn  Prod- 
ucts Refining  Company  the  starch  and  "refinery  mud"  used  in  this  investigation, 
and  for  his  valuable  suggestions  concerning  the  determination  of  the  amount  of  com- 
bined fat. 


12 

Per  cent. 

Moisture 29.5 

Protein 10.9 

Ash 35 

Red.  sugar 2.4 

Fat 48.8 

Remainder Undetermined 

The  fat  content  is  high  because  included  in  this  are  other  substances, 
especially  iron  soaps  which  dissolve  with  the  fat,  but  these  probably  do 
not  amount  to  more  than  2.0%  of  the  total  extractable  matter. 

When  the  fat  is  first  recovered  it  is  a  light  yellow  mass  with  an  odor 
suggesting  furfural,  but  on  standing  exposed  to  air  it  soon  darkens  and 
becomes  gummy.  After  a  few  days  the  odor  is  typical  of  oleic  acid  which 
has  been  exposed  to  the  air  for  a  time. 

Recovery  of  Fat  from  the  Refinery  Mud. — A  kilo  of  this  mud  was 
thoroughly  mixed  with  about  a  liter  of  low  boiling  petroleum  ether  and 
then  poured  into  a  2 -liter  round-bottom,  short-neck  flask  connected  with 
a  spiral  condenser.  The  flask  was  heated  on  a  water  bath  while  vacuum 
was  applied  to  the  system,  water  and  petroleum  ether  distilled  over  to- 
gether and  separated  into  2  layers  in  the  receiver.  The  ether  layer  was 
removed  and  returned  to  the  flask.  On  distilling  over  the  ether  repeatedly 
the  water  was  removed  from  the  mud  without  undue  exposure  of  the  fat 
to  the  air  and  in  addition  the  drying  was  uniform  throughout  the  mass. 
Finally  the  last  suspension  of  the  starch  in  the  petroleum  ether  was  filtered 
through  a  Biichner  funnel  under  suction  and  the  ether  distilled  off,  leaving 
the  crude  fat  in  a  dark  brown  mass  whose  surface  -showed,  as  the  fat  be- 
came cold,  a  peculiar  crystalline  structure.  This  crude  fat  was  dissolved 
in  boiling  alcohol  and  repeatedly  treated  with  bone  black  and  then  re- 
crystallized  from  alcohol  until,  on  cooling,  a  perfectly  white  crystalline 
fatty  acid  separated.  Several  more  crystallizations  were  made  until  the 
product  showed  no  iodine  number.  A  weighed  sample^of  this  final  product 
was  dissolved  in  neutral  alcohol  and  titrated  with  standard  alkali. 

Subs.,  0.2788,  0.5000:  NaOH  required,  0.0437,  0.0739.     Found:  Mol.  wt.  (mono- 
basic acid):  254.8,  256.5. 

The  iodine  value  for  this  material  was  zero. 

A  silver  soap  was  prepared  by  slightly  over-neutralizing  the  free  fatty 
acid  with  ammonia  and  then  adding  silver  nitrate  solution  until  no  further 
precipitate  formed.     The  precipitate  was  filtered  off,  washed  free  of  salts, 
dried,  and  a  weighed  portion  ignited  in  a  tared  porcelain  crucible.     A 
residue  of  silver  resulted  which  was  weighed  and  from  this  value  the 
molecular  weight  of  the  substance  calculated  as  a  monobasic  acid. 
Subs.,  0.212:  Ag,  0.006 1. 
Calc.  for  palmitic  acid:  256.6.    Found:  268.3. 

The  acid,  after  remaining  in  an  ice  box  for  several  hours,  melted  at 


13 

6 1. 6°,  with  a  rate  of  heating  of  about  i°  per  minute.  Lewkowitsch  gives 
62.6°  as  the  melting  point  for  palmitic  acid.  The  anilide  of  our  acid 
melted  at  90.2°  which  is  the  melting  point  given  for  the  anilide  of  palmitic 
acid.  The  substance,  upon  combustion,  agrees  in  composition  with  that 
of  palmitic  acid. 

Calc.  for  palmitic  acid:  C,  74.92;  H,  12.54.     Found:  C,  75.00;  H,  12.36. 

The  freshly  isolated  fat  mentioned  in  II,  however,  has  a  melting  point 
between  38°  and  43°,  an  acid  number  of  186,  and  an  iodine  number  of 
about  92,  while  palmitic  acid,  as  pointed  out  above,  has  a  melting  point  of 
62°,  an  acid  number  of  219,  and  a  practically  zero  iodine  number.  There 
is,  therefore,  present  in  the  fat,  liberated  in  the  hydrolysis  of  the  purified 
starch,  besides  the  palmitic  acid  an  unsaturated  constituent  of  relatively 
low  acidity. 

In  order  to  get  some  insight  into  the  nature  of  this  other  constituent, 
the  lead  salts  were  made  according  to  the  usual  method  and  the  ether 
soluble  portion  (the  lead  salts  of  certain  unsaturated  acids  are  soluble 
in  ether)  decomposed  with  hydrogen  sulfide.  The  liberated  fatty  material 
was  liquid  at  ordinary  temperature  and  had  an  iodine  number  of  136.0. 
On  cooling,  some  solid  separated,  leaving  a  light  yellow  liquid.  The 
solid  was  removed  from  the  liquid  and  dried  between  filter  paper  and  was 
apparently  palmitic  acid,  since  it  melted  at  62°  and  had  an  acid  number  of 
202.  The  light  yellow  liquid  remaining  was  highly  unsaturated,  had  an 
iodine  value  of  136,  and  showed  practically  no  acidity.  It  was  insoluble  in 
cold  aqueous  alkali,  coagulating  as  a  gummy  mass,  showed  the  presence  of 
small  amounts  of  reducing  sugar  when  treated  with  Fehling's  solution, 
and  gave  no  test  for  nitrogen  by  the  sodium  fusion  method,  but  did  show 
the  presence  of  small  amount  of  phosphorus,  and  gave  no  residue  on 
ignition.  This  material  will  be  referred  to  again  as  X  in  the  latter  part 
of  the  article. 

IV.  Liberation  of  the  Fatty  Acids  at  the  Early  Stages. 
Erythrodextrin  of  the  Hydrolysis  of  the  Starch.  Hydrolysis  by 
Mineral  Acids. — Fifty  g.  of  the  purified  starch,  suspended  in  water,  was 
treated  with  3  M  hydrochloric  acid,  and  when  the  erythrodextrin  stage 
of  hydrolysis,  indicated  by  plum  color  with  iodine,  was  reached,  the  opera- 
tion was  arrested,  and  the  mixture  poured  into  an  excess  of  95%  alcohol. 
The  alcohol  caused  a  separation  of  the  dextrin,  which  was  removed  by 
filtering  and  then  washed  with  alcohol  and  ether.  The  combined  alcohol- 
ether  filtrates  and  washings  were  evaporated,  the  residue  remaining  ex- 
tracted with  ether  and  the  extract  evaporated,  weighed,  and  found  to 
amount  to  0.53%  of  the  original  starch.  The  dextrin  was  hydrolyzed 
and  the  "fat  by  hydrolysis"  from  this  was  found  to  be  negligible.  When 
it  is  recalled  that  the  "fat  by  hydrolysis"  from  the  original  starch  is  be- 


14 

tween  0.5  and  0.6%  (see  table  under  II)  it  becomes  evident  from  these 
results  that  practically  all  the  fat  is  liberated  in  the  early  stages  of  hy- 
drolysis of  the  starch  by  aqueous  acids.  By  repeating  the  above  pro- 
cedure with  various  concentrations  of  hydrochloric  and  nitric  acids  the 
same  results  were  obtained  except  that  the  lower  concentrations  of  acid 
seemed  to  favor  the  formation  of  insoluble  retrograded  starch  which  re- 
sisted further  hydrolytic  action.  It  was  found,  however,  that  a  short 
treatment  with  10%  aqueous  sodium  carbonate  solution  rendered  the 
retrograded  starch  soluble  so  that  acid  reagents  could  act  in  the  usual 
way  to  give  glucose. 

Diastatic  Hydrolysis. — The  starch  was  hydrolyzed  by  means  of 
diastase  also  instead  of  by  acid.  It  was  thought  that  possibly  the  fat 
might  be  liberated  at  a  later  stage  in  the  hydrolysis  than  the  erythro- 
dextrin  when  the  enzyme  was  used.  The  procedure  was  practically  the 
same  as  in  the  acid  hydrolysis  except  that  in  order  to  secure  optimum 
conditions  for  the  diastatic  action,  the  hydrogen  ion  concentration  was 
adjusted  according  to  the  directions  of  Sherman,  Thomas  and  Baldwin.1 
Two  experiments  were  carried  out  and  in  each  case  free  fat  was  obtained. 
In  one  case  the  hydrolysis  was  interrupted  at  the  erythrodextrin  stage  and 
the  alcohol-ether  residue  similar  to  that  described  for  acid  hydrolysis, 
amounted  to  0.19%.  In  the  other  case  the  hydrolysis  was  allowed  to 
continue  until  a  test  portion  gave  practically  no  color  with  iodine,  and 
then  the  liberated  fat  amounted  to  0.41%  of  the  original  starch.  It 
therefore  appears,  as  far  as  one  can  judge  by  means  of  the  iodine  color, 
that  at  least  some  fat  is  liberated  at  the  erythrodextrin  stage  when  starch 
is  hydrolyzed  by  the  diastase  just  as  was  observed  when  acid  was  the 
hydrolytic  agent. 

V.  Cleavage  of  Starch  into  Products  of  Relatively  High  Fat  Content. 

(A).  Hydrolysis  or  Cleavage  of  Starch  by  Low  Hydrogen-Ion  Con- 
centration in  the  Presence  of  Bacillus  Aceto-ethylicum. — One  hundred 
g.  portions  of  starch  were  gelatinized  by  stirring  into  2  liters  of  boiling 
water  containing  2  g.  of  ammonium  phosphate  and  0.5  g.  of  magnesium 
sulfate  as  nutriment  for  the  bacteria.2  Starch  purified  as  described  above 
formed  in  this  way  a  very  limpid  paste.3  The  last  traces  of  foreign 
material  such  as  cell  fragments  still  present,  settled  and  were  removed 
by  the  decantation  of  paste  through  a  layer  of  cotton.  The  decanted 
paste  was  then  placed  in  a  5 -liter  flask  and  the  hydrogen  ion  adjusted  to 
approximately  io~8  by  adding  acid  or  alkaji  until  a  test  sample  showed  no 
pink  color  with  phenolphthalein  and  no  yellow  with  phenol  red.4  The 

1  Sherman,  Thomas  and  Baldwin,  J.  Am.  Chem.  Soc.,  41,  231  (1919)- 

2  Schardinger,  Centr.  Bakt.,  22,  98  (1909). 

8  Small,  /.  Am.  Chem.  Soc.,  41,  113  (1919). 
4  Lubs  and  Clark,  /.  Bact.t  4,  107  (1919). 


15 

flask  was  then  stoppered  with  cotton,  and  the  contents  sterilized  at  15 
pounds  steam  pressure  for  one  hour,  cooled  to  40°  and  inoculated  with 
bacillus  aceto-ethylicum,1  and  allowed  to  remain  in  an  incubator  at  40° 
until  there  was  no  more  apparent  action.  A  very  limpid  liquid,  con- 
taining a  small  amount  of  insoluble  material,  resulted.  This  was  filtered 
and  the  residue  obtained  was  washed  with  hot  water  and  allowed  to  drain. 
The  residue  then  was  stirred  into  an  excess  of  absolute  alcohol,  the  alcohol 
filtered  off  and  the  material  dried  in  vaciw  at  40°.  In  this  way  a  gray 
powder  was  finally  obtained  which  was  digested  with  boiling  ether,  filtered 
through  a  Biichner  funnel  and  washed  with  several  small  portions  of  ether 
to  remove  any  fat  which  might  have  been  freed  in  the  fermentation. 
The  residue  thus  obtained  produced  a  plum  color  with  iodine  and  showed 
no  starch  granules  under  the  microscope. 

Three  such  residues,  ranging  in  weight  from  10  to  15  g.,  were  hydrolyzed 
by  acid  as  in  II  and  were  found  to  contain  still  a  considerable  quantity  of 
fat.  The  values  given  below  were  calculated  on  the  dry  weight  of  the 
residues. 

Fat  of  hydrolysis. 
No.  %. 

i 1.6 

2 3-8 

3 2.2 

(B).  Hydrolysis  or  Cleavage  of  Starch  by  Low  Hydrogen  Ion  Con- 
centration without  Addition  of  Aceto-ethylicum. — On  allowing  solu- 
tions, prepared  for  inoculation  as  above,  to  stand,  a  separation  into  layers 
was  noticed,  an  upper  clear  and  a  lower  turbid  one.  The  latter  was 
similar  in  appearance  to  retrograded  starch.  This  observation  led  to  the 
carrying  out  of  the  above  partial  hydrolysis  of  the  starch  without  the 
addition  of  the  salts  or  aceto-ethylicum,  the  procedure  otherwise  being 
identical  with  that  in  (A).  After  standing  for  about  6  hours  a  rather 
complete  settling  of  the  insoluble  portion  had  taken  place.  The  residue 
obtained  by  this  latter  method  differed  from  that  described  in  (A)  in  that 
it  was  more  voluminous  and  had  the  appearance  of  retrograded  starch. 
It  was  removed  by  filtration  and  treated  in  a  way  analogous  to  the  pro- 
cedure employed  for  the  residue  in  (A). 

Three  100  g.  portions  of  purified  starch  containing  0.52  g.  of  "fat  by 
hydrolysis"  were  analyzed  by  this  method. 

Residue  from  "Fat  by  hydrolysis" 

100  g.  of  starch  in  residue. 

Residue  No  G.  %. 

88  5-0  5-5 

89  8.1  3-6 

90  8.0  3-3 

1  The  authors  wish  to  thank  Dr.  J.  H.  Northrop  for  the  culture  of  the  Bacillus 
accto-ethylicum  and  directions  for  applying  it  to  starch.  This  is  the  bacteria  used  by 
Northrop,  Ashe  and  Morgan  for  the  production  of  acetone  from  starch  and  glucose 
See  /.  Ind.  Eng.  Chem.,  u,  723  (1919). 


i6 

A  sample  of  residue  No.  88  gave  on  ignition  an  alkaline  ash  of  0.28%, 
which  showed  the  presence  of  phosphorus  and  magnesium,  while  residue 
itself  showed  0.04%  of  phosphorus. 

Since  these  residues  contained  as  high  fat  content  as  those  described 
in  (A),  the  method  of  preparation  in  (B)  is  to  be  preferred  due  to  its  being 
simpler.  The  fat  content  of  the  residues  from  both  methods,  (A)  and 
(B),  being  about  6  times  that  of  the  original  starch,  this  appears  to  offer  an 
avenue  to  the  isolation  of  a  simpler  compound  and  information  concerning 
the  structure  of  the  fat-carbohydrate  linking.  Work  is  now  in  progress  on 
this  particular  phase  of  the  problem.  That  the  fat  in  the  residue  might  be 
occluded  in  the  residue,  is  very  improbable  on  account  of  the  method  em- 
ployed in  precipitation  of  the  residue  by  alcohol  and  ether,  both  of  which 
are  good  fat  solvents.  The  argument  that  the  residue  material  was  so 
insoluble  as  to  prevent  the  solvents  from  coming  into  contact  with  the 
fat  and  hence  the  latter  being  retained,  although  free,  in  the  residue,  can 
have  very  little  justification  in  the  light  of  the  following  experiment. 

Two  g.  of  residue  No.  90  was  refluxed  for  one  hour  with  20  cc.  of  o.i  M 
alkali.  This  treatment  gave  a  slightly  opalescent  liquid  similar  in  ap- 
pearance to  colloidal  starch.  On  pouring  the  liquid  into  an  excess  of 
alcohol  a  precipitate  was  formed  which  gave  a  blue  color  with  acid  iodine 
solution,  indicating  unchanged  starch.  The  precipitate  was  filtered, 
dried  and  extracted  with  boiling  alcohol  and  ether,  and  upon  analysis 
showed  1.9%  of  "fat  by  hydrolysis"  with  an  acid  number  of  175.7. 

By  this  alkali  treatment  the  residue  was  brought  into  solution  again, 
and  if  the  fat  had  been  free  fatty  acid  it  would  have  been  removed  com- 
pletely, yet  the  results  showed  that  only  1.4  of  the  3.3%  fat  present  in  the 
residue  was  removed.  The  reason  why  the  1.4%  was  removed  can  be 
explained  most  satisfactorily  by  assuming  that  the  fat  is  present  in  the 
original  residue  as  ester  and  that  under  the  conditions  of  the  above  experi- 
ment the  saponification  of  ester  by  the  alkali  was  not  complete. 

Fat  not  Accounted  for  in  Residue  Described  in  B. — It  is  evident  that 
only  about  l/2  of  the  total  combined  fat  in  the  original  starch  remains  as 
such  in  the  residue  No.  89.  The  aqueous  filtrate,  obtained  in  the  pre- 
paration of  this  residue,  was  extracted  with  ether  to  make  sure  that  no 
free  fat  was  present,  and  subjected  to  acid  hydrolysis.  In  this  way,  the 
filtrate  was  found  to  contain  0.085  g.  of  fat  still  combined  with  the  soluble 
starch  contained  in  the  filtrate.  Subtracting  the  sum  of  the  combined 
fat  held  in  the  residue  and  aqueous  filtrate,  0.292  +  0.085  or  0.38  g., 
from  0.56  g.,  the  "fat  by  hydrolysis"  in  the  original  100  g.  of  starch,  gives 
o.i 8  g.  of  fat  which  must  have  been  liberated  in  the  process  and  lost  by 
being  extracted  by  the  alcohol-ether  treatment.  Qualitative  test  of  the 
filtrate  with  Fehling's  solution  showed  reducing  sugar,  and  iodine  test 
indicated  erythrodextrin. 


17 

Decomposition  of  the  Starch  by  Alcohol  Solution  of  Hydrogen  Chloride. 

— In  the  preparation  of  some  methyl  glucoside  in  this  laboratory  by  the 
method  described  by  Fischer,1  maize  starch  was  heated  in  an  autoclave 
with  an  absolute  methyl  alcohol  solution  of  hydrogen  chloride.  An  insoluble 
residue,  similar  in  appearance  to  the  sediments  described  above,  together 
with  an  oily  liquid  floating  upon  the  surface  of  the  alcoholic  solution, 
were  formed.  This  residue  and  the  oily  liquid  were  examined  in  the  hope 
that  further  light  might  be  cast  upon  the  way  the  fat  is  combined  within 
the  starch. 

Since  the  reaction  of  the  starch  with  the  alcoholic  hydrogen  chloride 
had  taken  place  in  a  copper  autoclave,  there  was  some  contamination 
with  copper  salts.  It  was  necessary  to  remove  the  latter  by  washing 
with  glacial  acetic  acid  before  the  residue  could  be  examined.  The 
residue  showed  microscopically  only  flocks  of  amorphous  material,  which 
produced  a  red  color  with  iodine  similar  to  that  from  erythrodextrin. 
The  "fat  by  hydrolysis"  in  this  residue  was  0.56%  which  is  practically 
the  same  as  in  the  original  starch  (see  table  under  II).  It  is  of  interest  in 
this  connection  to  compare  these  results  with  those  obtained  in  the  aqueous 
acid  hydrolysis.  Although  the  iodine  color  of  the  various  cleavage 
products  of  starch  can  not  be  considered  absolutely  reliable,  it  serves  to 
show  roughly  that  a  change  has  taken  place  in  the  starch  molecule.  Com- 
paring this  color  reaction  of  the  material  from  the  aqueous  hydrolysis 
with  that  obtained  from  the  alcoholic-hydrochloric  acid  treatment,  the 
following  facts  can  be  set  down. 

In  the  aqueous  hydrolysis  (see  Part  III)  as  soon  as  there  was  any  change 
in  the  starch  complex,  noted  by  the  iodine  test,  the  entire  amount  of 
bound  fat  was  liberated.  In  the  alcoholic  cleavage,  although  apparently 
all  the  starch  had  undergone  a  change  as  shown  by  the  red  color  with 
iodine  and  by  the  amorphous  condition  of  the  residue  as  revealed  by  the 
microscope,  the  erythrodextrin-like  residue  retained  the  same  quota  of 
fat  as  is  present  in  the  original  starch.  It  would  seem,  therefore,  from 
these  rather  limited  data  that  the  cleavage  of  the  starch  molecule  takes 
place  at  different  linkings  according  to  the  method  used,  and  furthermore 
it  serves  as  additional  evidence  that  the  fat  is  an  inherent  part  of  the 
starch  molecule. 

When  the  above  alcoholic  nitrate  is  evaporated  and  extracted  with 
ether  a  dark  brown  oil  is  obtained.  It  was  non-acidic,  decolorized 
bromine,  was  insoluble  in  cold  alkali  but  on  warming  practically  all  of  it 
dissolved  and  gave  a  soapy  solution  which  reduced  Fehling's  solution. 
Apparently  this  substance  is  the  unsaturated  constituent  "X,"  mentioned 
in  Part  III. 

^Fischer,  Ber.,  28,  1151  (1895). 


i8 

Treatment  of  Residue  Containing  Relatively  a  Large  Amount  of  Fat, 
with  Absolute  Alcohol-hydrogen  Chloride. — Since  the  residue  from  (A) 
and  (B)  still  contained  carbohydrate  very  similar  to  starch  itself,  and 
cleavage  with  hydrolytic  reagents  and  the  bacillus  was  apparently  im- 
possible, it  occurred  to  us  that  treatment  with  alcoholic  hydrogen  chloride 
might  offer  a  means  of  breaking  down  this  still  complex  compound  to  a 
simpler  one.  Accordingly  the  following  experiment  was  made. 

Five  g.  of  residue  No.  99*  was  refluxed  for  50  hours  with  160  cc.  of 
absolute  alcohol  containing  1.971  g.  of  hydrogen  chloride.  After  this 
treatment  there  remained  2.24  g.  of  apparently  unattacked  starch-like 
residue  which  gave  a  red  color  when  tested  with  iodine  instead  of  the 
purple  color  given  by  the  original  sample.  Analysis  of  this  sample,  after 
washing  with  ether,  showed  3.2%  of  "fat  by  hydrolysis"  referred  to  the 
weight  of  the  dry  product,  a  fat  content  essentially  the  same  as  that  of  the 
original  sample  No.  89  (see  table)  and  therefore  indicating  that  only 
about  half  of  the  original  sample  had  been  affected  by  the  treatment. 
On  partial  evaporation  of  the  alcoholic  solution  containing  the  glucoside, 
there  appeared  on  the  surface  small  droplets  of  dark  brown  oil.  These 
were  removed  by  solution  in  ether;  and  on  evaporation  of  the  ether  a 
product  remained  which  was  similar  in  properties  to  the  unsaturated 
liquid  called  "X"  described  above. 

VI.  The  Fatty  Acid  is  Combined  Indirectly  to  the  Starch. 

(A)  Saponification  of  Fat  Liberated  by  Acid  Hydrolysis. — A  sample 
of  0.3058  g.  of -fat  liberated  by  aqueous  acid  hydrolysis  was  refluxed  for 
one  hour  with  5  cc.  of  M  alcoholic  sodium  hydroxide,  while  as  a  blank 
analysis  5  cc.  of  sodium  hydroxide  was  given  exactly  the  same  treat- 
ment.    After  refluxing,  the  flasks  were  cooled  and  titrated  back  with 
0.05  M  sulfuric  acid. 

Cc.  OF  0.05  M  SULFURIC  ACID  REQUIRED. 

Blank 48 . 9 

Sample 38.0 

Difference 10. 9 

This  was  equivalent  to  a  saponification  number  of  199.5.  Another 
portion  of  same  sample  gave  an  acid  number  of  182.3,  while  that  of 
palmitic  acid  is  219.1. 

(B)  Saponification   of  Fat  Liberated   by  Diastatic   Hydrolysis.— A 
sample  of  0.1290  g.  of  fat  liberated  by  diastatic  hydrolysis  was  dissolved 
in  petroleum  ether  and  10  cc.  of  o.i  M  alcoholic  sodium  hydroxide  added. 
The  mixture  protected  from  carbon  dioxide  by  a  soda-lime  tube  was 
allowed  to  remain  at  room  temperature  for  18  hours.     Back  titration 
of  the  excess  alkali  required  5.1  cc.  of  0.05  M  sulfuric  acid,  indicating 
therefore  a  saponification  number  of  212.7.    The  acid  number  of  the  same 
sample  was  147.8. 


19 

It  is  evident  that  the  fat  from  both  methods  of  hydrolysis  of  the  starch 
contains  combined  fatty  acid  or  an  ester.  This  ester  has  suffered  more 
hydrolysis  when  acid  instead  of  diastase  was  used  in  decomposing  the 
starch.  In  the  light  of  these  data,  very  likely  the  unsaturated  con- 
stituent present  in  the  fat,  labeled  "X,"  is  combined  with  the  palmitic 
acid  and  accounts  for  the  presence  of  the  ester. 

The  fact  that  the  palmitic  acid,  which  is  a  mono-basic  acid,  is  linked  as 
an  ester  to  the  unsaturated  compound  ("X")  precludes  the  possibility  of 
direct  carbohydrate  fatty  acid  union,  and  since  it  has  been  shown  that  the 
entire  fatty  material  (fatty  acid  and  unsaturated  substance  "X")  is  com- 
bined with  the  carbohydrate  and  only  liberated  by  hydrolysis,  the  sub- 
stance "X"  must  serve  as  a  connecting  link  between  the  acid  and  the 
carbohydrate. 

Starches  from  other  sources  than  corn  also  contained  combined  fat. 

Samples  of  various  representative  starches  were  purified  as  described 
under  Part  I  and  the  "fat  by  hydrolysis"  determined.  In  the  following 
table  are  enumerated  the  results  of  these  analyses.  Fifty  g.  samples 
were  used,  except  in  the  case  of  potato  starch. 


Starch. 

Corn  

"Fat  by 
hydrolysis." 

o  61 

Acid  No. 

182  s 

Iodine  No. 
O2    5 

Rice  ...    . 

o  8^ 

28^    4. 

84    7 

Saeo... 

O    II 

151  o 

Cassava 

O    12 

1  68  o 

Potato  (200  g.  sample)  

O  O4. 

IOO   4. 

Horse  chestnut  .  . 

o.«%6 

21  .O 

From  the  above  results  it  is  evident  that  corn  starch  is  not  unique  in 
having  combined  fat,  but  that  starches  of  widely  differing  genera  also 
contain  combined  fatty  material. 

We  are  greatly  indebted  to  the  Harriman  Research  Laboratory,  New 
York  City  for  grants  in  the  aid  of  this  investigation 


VITA. 

Theodore  Clinton  Taylor  was  born  in  Mount  Vernon,  New  York, 
October  i,  1891.  He  entered  Columbia  University,  School  of  Applied 
Science,  in  the  Fall  of  1911  and  was  graduated  with  the  degree  of  Chemical 
Engineer  in  1915.  From  that  time  until  the  Fall  of  1917  he  was  employed 
as  chemist  for  the  Corn  Products  Refining  Company,  when  he  returned 
to  the  University  and  commenced  the  studies  required  for  the  degree  of 
Doctor  of  Philosophy.  In  June  1918  he  received  the  degree  of  Master  of 
Arts,  after  which  he  left  the  University  to  go  into  National  Service.  He 
returned  to  the  University  in  January  1919  and  again  resumed  the  studies 
for  the  doctorate.  From  January  1 9 1 9  until  October  1919  he  was  Harri- 
man  Research  Assistant,  when  he  was  appointed  Lecturer  in  Organic 
Chemistry  in  Columbia  College. 


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4  days  prior  to  due  date 


DUE  AS  STAMPED  BELOW 


SEP  2  4  2004 


DD20  15M  4-02 


'pftl.JMi.2V"08 


£51732 


UNIVERSITY  OF  CALIFORNIA  LIBRARY 


